Development of a bacteriophage-based biopesticide for fire blight

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Abstract

Fire blight is an economically important disease of apples and pears that is caused by the
bacterium Erwinia amylovora. Control of the disease depends on limiting primaly blosson1
infection in the spring, and rapidly removing infected tissue. The possibility of using phages to
control E.amylovora populations has been suggested, but previous studies have. failed to show
high treatment efficacies. This work describes the development of a phage-based biopesticide
that controls E. amylovora populations under field conditions, and significantly reduces the
incidence of fire blight.
This work reports the first use ofPantoea agglomerans, a non-pathogenic relative ofE.
amylovora, as a carrier for E. amylovora.phages. Its role is to support a replicating population of
these phages on blossom surfaces during the period when the flowers are most susceptible to
infection. Seven phages and one carrier isolate were selected for field trials from existing
collections of 56 E. amylovora phages and 249 epiphytic orchard bacteria. Selection of the .
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phages and carrier was based on characteristics relevant to the production and field perfonnance
of a biopesticide: host range, genetic diversity, growth under the conditions of large-scale
production, and the ability to prevent E. amylovora from infecting pear blossoms. In planta
assays showed that both the phages and the carrier make significant contributions to reducirig the
development of fire blight symptoms in pear blossoms.
Field-scale phage production and purification methods were developed based on the
growth characteristics of the phages and bacteria in liquid culture, and on the survival of phages
in various liquid media.
Six of twelve phage-carrier biopesticide treatments caused statistically signiflcant reductions in disease incidence during orchard trials. Multiplex real-time PCR was used to
simultaneously monitor the phage, carrier, and pathogen populations over the course of selected
treatments. In all cases. the observed population dynamics of the biocontrol agents and the
pathogen were consistent with the success or failure of each treatment to control disease
incidence. In treatments exhibiting a significantly reduced incidel1ce of fire blight, the average
blossom population ofE.amylovora had been reduced to pre-experiment epiphytic levels. In
successful treatments the phages grew on the P. agglomerans carrier for 2 to 3 d after treatment
application. The phages then grew preferentially on the pathogen, once it was introduced into this
blossom ecosystem. The efficacy of the successful phage-based treatnlents was statistically
similar to that of streptomycin, which is the most effective bactericide currently available for fire
blight prevention.
The in planta behaviour ofE. amylovora was compared to that ofErwinia pyrifoliae, a
closely related species that causes fire blight-like synlptoms on pears in southeast Asia. Duplex
real-time PCR was used to monitor the population dynamics of both species on single blossonls.
E. amylovora exhibited a greater competitive fitness on Bartlett pear blossoms than E. pyrifoliae.
The genome ofErwinia phage <l>Ea21-4 was sequenced and annotated. Most of the 8-4.7
kB genome is substantially different from previously described sequences, though some regions
are notably similar to Salmonella phage Felix 01 . Putative functions were assigned to
approximately 30% of the predicted open reading frames based on amino acid sequence
comparisons and N-terminal sequencing of structural proteins.